PDBsum entry 1l9r

Oxidoreductase

PDB id: 1l9r

Contents

Protein chains

336 a.a. *

Ligands

NO2 ×3

Metals

_CU ×6

Waters ×1473

* Residue conservation analysis

PDB id:

1l9r

Name:

Oxidoreductase

Title:

Crystal structure of the i257m variant of the copper-containing nitrite reductase from alcaligenes faecalis s-6

Structure:

Copper-containing nitrite reductase. Chain: a, b, c. Fragment: residues 40-376. Synonym: cu-nir. Engineered: yes. Mutation: yes

Source:

Alcaligenes faecalis. Organism_taxid: 511. Gene: nirk. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Trimer (from PQS)

Resolution:

1.78Å R-factor: 0.154 R-free: 0.197

Authors:

M.J.Boulanger,M.E.P.Murphy

Key ref:

M.J.Boulanger and M.E.Murphy (2003). Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine. Protein Sci, 12, 248-256. PubMed id: 12538888 DOI: 10.1110/ps.0224503

Date:

26-Mar-02 Release date: 04-Feb-03

Protein chains

P38501  (NIR_ALCFA) -  Copper-containing nitrite reductase from Alcaligenes faecalis

Seq: 376 a.a.

Struc: 336 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.1.7.2.1 - nitrite reductase (NO-forming).
• Reaction: nitric oxide + Fe(III)-[cytochrome c] + H2O = Fe(II)-[cytochrome c] + nitrite + 2 H⁺

nitric oxide + Fe(III)-[cytochrome c] + H2O = Fe(II)-[cytochrome c] + nitrite + 2 × H(+) (Bound ligand (Het Group name = NO2) corresponds exactly)

• Cofactor: Cu cation or Fe cation; FAD

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1110/ps.0224503

Protein Sci 12:248-256 (2003)

PubMed id: 12538888

Directing the mode of nitrite binding to a copper-containing nitrite reductase from Alcaligenes faecalis S-6: characterization of an active site isoleucine.

M.J.Boulanger, M.E.Murphy.

ABSTRACT

Unlike the heme cd(1)-based nitrite reductase enzymes, the molecular mechanism of copper-containing nitrite reductases remains controversial. A key source of controversy is the productive binding mode of nitrite in the active site. To identify and characterize the molecular determinants associated with nitrite binding, we applied a combinatorial mutagenesis approach to generate a small library of six variants at position 257 in nitrite reductase from Alcaligenes faecalis S-6. The activities of these six variants span nearly two orders of magnitude with one variant, I257V, the only observed natural substitution for Ile257, showing greater activity than the native enzyme. High-resolution (> 1.8 A) nitrite-soaked crystal structures of these variants display different modes of nitrite binding that correlate well with the altered activities. These studies identify for the first time that the highly conserved Ile257 in the native enzyme is a key molecular determinant in directing a catalytically competent mode of nitrite binding in the active site. The O-coordinate bidentate binding mode of nitrite observed in native and mutant forms with high activity supports a catalytic model distinct from the heme cd(1) NiRs. (The atomic coordinates for I257V[NO(2)(-)], I257L[NO(2)(-)], I257A[NO(2)(-)], AfNiR have been deposited in the Protein Data Bank [PDB identification codes are listed in Table 2].)

Selected figure(s)

Figure 1.
Figure 1. Crystal structures of the six nitrite-soaked I257 variants in the upper six panels. Hydrogen bonds are shown as dashed gray lines with ligand bonds drawn in solid, dark gray lines. Water molecules are drawn as aquamarine spheres. Copper atoms are gray; nitrogen atoms are dark blue, oxygen atoms are red, and sulfur atoms are yellow. The backbone of monomers B and C are shown in burgundy and in teal, respectively. Bonds of the nitrite molecule bound in the active site are white. Omit Fo-Fc electron density maps are contoured at 4 and drawn as a green wire mesh. Panels A and B depict the multiple conformations of the nitrite bound in the active site of the Ile257 variants and including nitrite bound to the oxidized D98N (black atoms) and H255N (gray atoms) crystal structures (Boulanger and Murphy 2001). With the exception of the purple nitrite molecules in panel B, nitrite molecules are blue to red with increasing specific activity: Red, I257V[NO[2]^-]; orange, native NiR from Alcaligenes faecalis S-6; light orange, I257L[NO[2]^-]; yellow, I257M[NO[2]^-]; green, I257A[NO[2]^-]; cyan, I257G[NO[2]^-]; blue, I257T[NO[2]^-].

The above figure is reprinted by permission from the Protein Society: Protein Sci (2003, 12, 248-256) copyright 2003.

Figure was selected by an automated process.